def do_resequencing(self, data_file, amp_analysis_records, min_length,
whitelist_file):
log.info('Resequencing supplied Amplicon Analysis sequences')
output_dir = self.get_output_folder("amp_analysis_resequencing")
# Convert the raw data file into a BaxH5 fofn for use downstream
# and create appropriate reader for local access
bash5 = get_bash5_reader(data_file)
baxh5_file = os.path.join(self.output, 'baxh5.fofn')
create_baxh5_fofn(data_file, baxh5_file)
# Extract any consensus sequences associated with this barcode
log.info('Identified {0} consensus sequences to resequence'.format(
len(amp_analysis_records)))
reference_file = os.path.join(output_dir, 'reference.fasta')
write_records(amp_analysis_records, reference_file)
# Resequence the selected consensus sequences with the selected ZMWs
self.resequencer(baxh5_file,
whitelist_file,
reference_file,
output=output_dir,
min_length=min_length)
log.info(
"Finished resequencing supplied Amplicon Analysis sequences\n")
def __call__(self,
amp_analysis,
data_file,
barcode_file,
barcode_string=None,
min_snr=None,
min_length=None):
log.info(
"Beginning Amplicon Analysis resequencing workflow for {0}".format(
amp_analysis))
# Pick or create a single file from AA and read it
amp_analysis_file = get_input_file(amp_analysis)
amp_analysis_records = list(AmpliconAnalysisReader(amp_analysis_file))
# Convert the raw data file into a BaxH5 fofn for use downstream
# and create appropriate reader for local access
bash5 = get_bash5_reader(data_file)
baxh5_file = os.path.join(self.output, 'baxh5.fofn')
create_baxh5_fofn(data_file, baxh5_file)
# Create a Reader for the Barcode data and find the overlap with any
# barcodes specified by the user
bc_reader = get_barcode_reader(barcode_file)
bc_list = get_barcodes(bc_reader, barcode_string)
for i, bc in enumerate(bc_list):
log.info('Resequencing Barcode {0} (#{1} of {2})'.format(
bc, i + 1, len(bc_list)))
output_dir = self.get_output_folder(bc)
# Extract any consensus sequences associated with this barcode
record_list = [r for r in amp_analysis_records if r.barcode == bc]
log.info(
'Identified {0} consensus sequences for Barcode {1}'.format(
len(record_list), bc))
filtered_records = [r for r in record_list if r.num_reads >= 20]
unique_records = get_unique_records(filtered_records)
fraction = 100 * round(
len(unique_records) / float(len(record_list)), 3)
log.info(
'{0} of {1} ({2}%) consensus sequences passed all filters'.
format(len(unique_records), len(record_list), fraction))
reference_file = os.path.join(output_dir, 'reference.fasta')
write_records(unique_records, reference_file)
# Identify all high-quality, barcode-specific ZMWs and write them to file
zmw_list = get_barcode_zmws(bc_reader, bc)
zmw_list = filter_zmw_list(bash5, zmw_list, min_snr=min_snr)
whitelist_file = os.path.join(output_dir, 'whitelist.txt')
write_zmw_whitelist(bash5, zmw_list, whitelist_file)
# Resequence the selected consensus sequences with the selected ZMWs
self.resequencer(baxh5_file,
whitelist_file,
reference_file,
output=output_dir,
min_length=min_length)
log.info("Finished resequencing Barcode {0}\n".format(bc))
def _create_baxh5_fofn( input_file, output ):
"""
Convert any BasH5 input files to BaxH5 to avoid file-type problems
"""
log.info('Creating FOFN of Bax.H5 files')
baxh5_fofn = os.path.join( output, 'baxh5.fofn' )
if valid_file( baxh5_fofn ):
log.info("Using existing Bax.H5 FOFN file")
return baxh5_fofn
log.info("No existing Bax.H5 fofn found")
create_baxh5_fofn( input_file, baxh5_fofn )
check_output_file( baxh5_fofn )
log.info('Finished writing Bax.H5 fofn file\n')
return baxh5_fofn
def _create_baxh5_fofn(input_file, output):
"""
Convert any BasH5 input files to BaxH5 to avoid file-type problems
"""
log.info('Creating FOFN of Bax.H5 files')
baxh5_fofn = os.path.join(output, 'baxh5.fofn')
if valid_file(baxh5_fofn):
log.info("Using existing Bax.H5 FOFN file")
return baxh5_fofn
log.info("No existing Bax.H5 fofn found")
create_baxh5_fofn(input_file, baxh5_fofn)
check_output_file(baxh5_fofn)
log.info('Finished writing Bax.H5 fofn file\n')
return baxh5_fofn
def __call__(self, amp_analysis, data_file, barcode_file, barcode_string=None, min_snr=None, min_length=None):
log.info("Beginning Amplicon Analysis resequencing workflow for {0}".format(amp_analysis))
# Pick or create a single file from AA and read it
amp_analysis_file = get_input_file( amp_analysis )
amp_analysis_records = list(AmpliconAnalysisReader(amp_analysis_file))
# Convert the raw data file into a BaxH5 fofn for use downstream
# and create appropriate reader for local access
bash5 = get_bash5_reader( data_file )
baxh5_file = os.path.join( self.output, 'baxh5.fofn')
create_baxh5_fofn( data_file, baxh5_file )
# Create a Reader for the Barcode data and find the overlap with any
# barcodes specified by the user
bc_reader = get_barcode_reader( barcode_file )
bc_list = get_barcodes( bc_reader, barcode_string )
for i, bc in enumerate( bc_list ):
log.info('Resequencing Barcode {0} (#{1} of {2})'.format(bc, i+1, len(bc_list)))
output_dir = self.get_output_folder( bc )
# Extract any consensus sequences associated with this barcode
record_list = [r for r in amp_analysis_records if r.barcode == bc]
log.info('Identified {0} consensus sequences for Barcode {1}'.format(len(record_list), bc))
filtered_records = [r for r in record_list if r.num_reads >= 20]
unique_records = get_unique_records( filtered_records )
fraction = 100 * round(len(unique_records)/float(len(record_list)), 3)
log.info('{0} of {1} ({2}%) consensus sequences passed all filters'.format(len(unique_records),
len(record_list),
fraction))
reference_file = os.path.join( output_dir, 'reference.fasta' )
write_records( unique_records, reference_file )
# Identify all high-quality, barcode-specific ZMWs and write them to file
zmw_list = get_barcode_zmws( bc_reader, bc )
zmw_list = filter_zmw_list( bash5, zmw_list, min_snr=min_snr )
whitelist_file = os.path.join( output_dir, 'whitelist.txt' )
write_zmw_whitelist( bash5, zmw_list, whitelist_file )
# Resequence the selected consensus sequences with the selected ZMWs
self.resequencer( baxh5_file,
whitelist_file,
reference_file,
output=output_dir,
min_length=min_length )
log.info("Finished resequencing Barcode {0}\n".format( bc ))
def do_resequencing(self, data_file, amp_analysis_records, min_length, whitelist_file):
log.info("Resequencing supplied Amplicon Analysis sequences")
output_dir = self.get_output_folder("amp_analysis_resequencing")
# Convert the raw data file into a BaxH5 fofn for use downstream
# and create appropriate reader for local access
bash5 = get_bash5_reader(data_file)
baxh5_file = os.path.join(self.output, "baxh5.fofn")
create_baxh5_fofn(data_file, baxh5_file)
# Extract any consensus sequences associated with this barcode
log.info("Identified {0} consensus sequences to resequence".format(len(amp_analysis_records)))
reference_file = os.path.join(output_dir, "reference.fasta")
write_records(amp_analysis_records, reference_file)
# Resequence the selected consensus sequences with the selected ZMWs
self.resequencer(baxh5_file, whitelist_file, reference_file, output=output_dir, min_length=min_length)
log.info("Finished resequencing supplied Amplicon Analysis sequences\n")
def do_barcoded_resequencing(
self, data_file, amp_analysis_records, barcode_file, barcode_string, min_snr, min_length
):
# Convert the raw data file into a BaxH5 fofn for use downstream
# and create appropriate reader for local access
bash5 = get_bash5_reader(data_file)
baxh5_file = os.path.join(self.output, "baxh5.fofn")
create_baxh5_fofn(data_file, baxh5_file)
# Create a Reader for the Barcode data and find the overlap with any
# barcodes specified by the user
bc_reader = get_barcode_reader(barcode_file)
bc_list = get_barcodes(bc_reader, barcode_string)
for i, bc in enumerate(bc_list):
log.info("Resequencing Barcode {0} (#{1} of {2})".format(bc, i + 1, len(bc_list)))
output_dir = self.get_output_folder(bc)
# Extract any consensus sequences associated with this barcode
record_list = [r for r in amp_analysis_records if r.barcode == bc]
log.info("Identified {0} consensus sequences for Barcode {1}".format(len(record_list), bc))
filtered_records = [r for r in record_list if r.num_reads >= 20]
unique_records = get_unique_records(filtered_records)
fraction = 100 * round(len(unique_records) / float(len(record_list)), 3)
log.info(
"{0} of {1} ({2}%) consensus sequences passed all filters".format(
len(unique_records), len(record_list), fraction
)
)
reference_file = os.path.join(output_dir, "reference.fasta")
write_records(unique_records, reference_file)
# Identify all high-quality, barcode-specific ZMWs and write them to file
zmw_list = get_barcode_zmws(bc_reader, bc)
zmw_list = filter_zmw_list(bash5, zmw_list, min_snr=min_snr)
whitelist_file = os.path.join(output_dir, "whitelist.txt")
write_zmw_whitelist(bash5, zmw_list, whitelist_file)
# Resequence the selected consensus sequences with the selected ZMWs
self.resequencer(baxh5_file, whitelist_file, reference_file, output=output_dir, min_length=min_length)
log.info("Finished resequencing Barcode {0}\n".format(bc))
